miR-99a通过下调RRM2抑制人子宫颈癌HeLa细胞系增殖、迁移和侵袭  

miR-99a inhibits the proliferation,migration and invasion of cervical cancer HeLa cell line through down-regulating RRM2

作　　者：贺春花[1] 刘冷[1] 张小柳[1] HE Chun-hua;LIU Leng;ZHANG Xiao-liu(Department of Gynecology,Jingmen No.1 People's Hospital,Jingmen 448000,China)

机构地区：[1]荆门市第一人民医院妇科,湖北荆门448000

出　　处：《基础医学与临床》2022年第11期1737-1743,共7页Basic and Clinical Medicine

基　　金：荆门市科学技术研究与开发计划(2019YDKY049)。

摘　　要：目的探讨miR-99a与核糖核苷酸还原酶小亚基M2(RRM2)的靶向关系,并分析二者对人子宫颈癌(CC)HeLa细胞系增殖、侵袭及迁移的影响。方法将HeLa细胞,分为对照组(control)、阴性对照组(inhibitor-NC)、抑制miR-99a表达组(miR-99a-inhibitor)、共转染组(RRM2-siRNA+miR-99a-inhibitor)。MTT法、平板集落实验检测细胞增殖;划痕实验、Transwell小室法检测细胞迁移、侵袭;Western blot检测RRM2、细胞增殖核抗原(PCNA)、E-钙黏附蛋白(E-cadherin)、N-钙黏附蛋白(N-cadherin)、波形蛋白(vimentin)蛋白表达;免疫荧光法检测E-cadherin、N-cadherin、vimentin定位及表达;生物信息学及双荧光素酶报告基因实验验证miR-99a与RRM2靶向关系;RT-qPCR检测细胞中miR-99a、RRM2 mRNA水平。结果下调miR-99a表达,可增高HeLa细胞增殖率(100.00比128.86±4.25,P<0.05);上调集落形成率、划痕愈合率、侵袭细胞数;增加RRM2、PCNA、N-cadherin与vimentin蛋白表达,降低E-cadherin蛋白表达(P<0.05)。抑制RRM2表达能减轻下调miR-99a对HeLa细胞生物学行为的影响(P<0.05)。双荧光素酶报告基因实验及RT-qPCR证实miR-99a与RRM2存在靶向关系(P<0.05)。结论抑制miR-99a可能靶向上调RRM2表达并促进HeLa细胞增殖,有望成为CC新的潜在治疗靶点。Objective To investigate the targeting relationship between miR-99 a and ribonucleotide reductase subunit M2(RRM2)and to analyze their effects on proliferation,invasion and migration of cervical cancer(CC)-oringinated HeLa cell line.Methods HeLa cells were divided into control group,negative control group(inhibitor-NC),inhibition of miR-99 a expression group(miR-99 a-inhibitor)and co-transfection group(RRM2-siRNA+miR-99 a-inhibitor).MTT assay and plate colony experiment were used to detect the proliferation ability of HeLa cells.Scratch test and Transwell test were used to detect cell migration and invasion ability respectively.Western blot was used to detect protein expression of RRM2,proliferating cell nuclear antigen(PCNA),E-cadherin,N-cadherin andvimentin.Immunofluorescence method was used to detect the localization and expression of E-cadherin,N-cadherin and vimentin.Bioinformatics and dual-luciferase reporter gene experiments verified the targeting relationship between miR-99 a and RRM2.RT-qPCR were used to detect the expression levels of miR-99 a and RRM2 mRNA in HeLa cells in each group.Results Down-regulating the expression of miR-99 a increased proliferation of HeLa cells(100.00 vs.128.86±4.25,P<0.05).Up-regulated colony formation improved scratch healing rate,and increased the number of invasive cells and the expression of RRM2,PCNA,N-cadherin and vimentin protein,decreased E-cadherin protein expression(P<0.05).Inhibition of RRM2 expression reversed the effect of down-regulation of miR-99 a on the biological behavior of HeLa cells(P<0.05).Dual-luciferase reporter gene experiment and RT-qPCR confirmed that miR-99 a had a targeting relationship with RRM2(P<0.05).Conclusions Inhibition of miR-99 a may target at up-regulating the expression of RRM2 to promote the development of HeLa cells,which is expected to become a new potential therapeutic target for CC.

关 键 词：miR-99a 核糖核苷酸还原酶小亚基M2 宫颈癌细胞 增殖 迁移 

分 类 号：R737[医药卫生—肿瘤]